Bioprosthetic heart valves

ABSTRACT

The invention provides methods and materials related to heart valves and the treatment of valvular heart disease. Specifically, the invention provides non-murine heart valve cells and heart valve cusps as well as methods for making heart valves. The invention also provides methods and materials for (1) slowing heart valve degeneration, thrombosis, and calcification, (2) treating carcinoid heart disease, (3) identifying inhibitors of heart valve degeneration, thrombosis, and calcification, and (4) determining the safety of drugs.

BACKGROUND

[0001] 1. Technical Field

[0002] The invention relates to bioprosthetic heart valves and thetreatment of valvular heart disease.

[0003] 2. Background Information

[0004] The heart is a hollow, muscular organ that circulates bloodthroughout an animal's body by contracting rhythmically. In mammals, theheart has four-chambers situated such that the right atrium andventricle are completely separated from the left atrium and ventricle.Normally, blood flows from systemic veins to the right atrium, and thento the right ventricle from which it is driven to the lungs via thepulmonary artery. Upon return from the lungs, the blood enters the leftatrium, and then flows to the left ventricle from which it is driveninto the systematic arteries.

[0005] Four main heart valves prevent the backflow of blood during therhythmic contractions: the tricuspid, pulmonary, mitral, and aorticvalves. The tricuspid valve separates the right atrium and rightventricle, the pulmonary valve separates the right atrium and pulmonaryartery, the mitral valve separates the left atrium and left ventricle,and the aortic valve separates the left ventricle and aorta. Generally,patients having an abnormality of a heart valve are characterized ashaving valvular heart disease.

[0006] A heart valve can malfunction either by failing to open properly(stenosis) or by leaking (regurgitation). For example, a patient with amalfunctioning aortic valve can be diagnosed with either aortic valvestenosis or aortic valve regurgitation. In either case, valvereplacement by surgical means is a possible treatment. Replacementvalves can be autografts, allografts, or xenografts as well asmechanical valves or valves made partly from pig valves. Interestingly,cryopreserved allografts remain viable within the recipient patient formany years after transplantation. Unfortunately, replacement valves aresusceptible to problems such as degeneration, thrombosis, andcalcification.

SUMMARY

[0007] The invention involves methods and materials related to heartvalves and the treatment of valvular heart disease. Specifically, theinvention provides heart valve cells and heart valve cusps as well asmethods for making heart valves. The invention also provides methods andmaterials for (1) slowing heart valve degeneration, thrombosis, andcalcification, (2) treating carcinoid heart disease, (3) identifyinginhibitors of heart valve degeneration, thrombosis, and calcification,and (4) determining the safety of drugs.

[0008] The invention is based on the discovery that heart valve cellsexpressing a polypeptide having nitric oxide synthase activity do notexhibit characteristics indicative of heart valve calcification anddegeneration. Specifically, heart valve cells containing nucleic acidthat encodes a polypeptide having nitric oxide synthase activityexhibit, in response to a valvular heart disease-promoting stimulus, adifferent extracellular matrix profile than that exhibited by similarcells lacking that nucleic acid. The molecular makeup of a heart valve'sextracellular matrix can be an important factor that determines thedegree of heart valve calcification and degeneration. For example,significant expression of osteopontin, a polypeptide abundant in bonematrix, can lead to significant tissue calcification. Heart valve cellscontaining nucleic acid that encodes a polypeptide having nitric oxidesynthase activity also exhibit, in response to a valvular heartdisease-promoting stimulus, less proliferation and apoptosis than theproliferation and apoptosi exhibited by similar cells lacking thatnucleic acid. The level of heart valve cell proliferation and apoptosiscan correlate with the degree of heart valve calcification anddegeneration. For example, significant proliferation and apoptosis ofheart valve cells can lead to heart valve thickening, and thus heartvalve malfunction and degeneration. It is noted that tissue thickeningoccurs presumably because cell proliferation out paces apoptosis overtime. The heart valve cells and cusps described herein can be used tomake bioprosthetic heart valves having a reduced susceptibility orprolonged resistance to degeneration, thrombosis, and calcification.Clearly, such bioprosthetic heart valves would be useful in any type ofheart valve replacement procedure.

[0009] The invention also is based on the discovery that serotoninreceptor antagonists (e.g., 5HT_(1B) receptor antagonists) can inhibitserotonin-induced proliferation of heart valve cells from patientsdiagnosed with carcinoid heart disease. Thus, serotonin receptorantagonists can be used to treat carcinoid heart disease. Further, themethods and materials used to identify serotonin receptor antagonists asa treatment for carcinoid heart disease also can be used to identifydrugs for the treatment of other valvular heart diseases as well as testthe safety of any drug designed for human use. Clearly, identifying newvalvular heart disease treatments and determining drug safety in generalwill greatly improve overall human health care.

[0010] In general, one aspect of the invention features a non-murineheart valve cell (e.g., an endothelial cell or myocyte) containing anexogenous nucleic acid that encodes a polypeptide having nitric oxidesynthase activity (e.g., endothelial nitric oxide synthase).

[0011] In another embodiment, the invention features an isolated heartvalve cusp where a cell of the cusp contains an exogenous nucleic acidthat encodes a polypeptide having nitric oxide synthase activity (e.g.,endothelial nitric oxide synthase). The cell can be a porcine or humancell.

[0012] Another embodiment of the invention features a method for makinga bioprosthetic heart valve. The method includes obtaining a heart valvecusp and introducing nucleic acid into a cell of the cusp, where thenucleic acid encodes a polypeptide having nitric oxide synthase activity(e.g., endothelial nitric oxide synthase). The cell can be a porcine orhuman cell. The nucleic acid can be introduced into the cell viaadenoviral-mediated nucleic acid transfer. The nucleic acid can beintegrated into the genome of the cell. The method can include fixingthe cusp, and the fixation step can occur after the introduction step.The method can include freezing the cusp, and the freezing step canoccur after the introduction step.

[0013] In another aspect, the invention features a method for slowingthe degeneration of a heart valve within a non-murine mammal. The methodincludes introducing nucleic acid encoding a polypeptide having nitricoxide synthase activity into a cell of the heart valve such that thepolypeptide is expressed. The introduction step can be performed invitro. The heart valve can be an autograft, allograft, or xenograft. Themethod can include administering an inhibitor of hydroxymethylglutarylcoA reductase activity to the mammal. The inhibitor can be pravastatin,atorvastatin, simvastatin, or lovastatin.

[0014] In another embodiment, the invention features a method forslowing heart valve degeneration. The method includes identifying amammal at risk of developing heart valve degeneration, and administeringan inhibitor of hydroxymethylglutaryl coA reductase activity to themammal. The mammal can contain a heart valve replacement. The mammal canhave congenital valvular disease or bicuspid valvular disease.

[0015] Another aspect of the invention features a method for treatingcarcinoid heart disease in a mammal. The method includes administering aserotonin receptor antagonist to the mammal. The antagonist can bespecific for a 5HT_(1B) receptor. The antagonist can contain aβ-blocker. The antagonist can be pindolol.

[0016] Another aspect of the invention features a method for identifyingan inhibitor of heart valve degeneration. The method includes contactingheart valve cells with a stimulant such that the cells proliferate,contacting the cells with a test compound, and determining if the testcompound reduced the proliferation of the cells, where the reduction ofproliferation indicates that the test compound is an inhibitor of heartvalve degeneration.

[0017] In another embodiment, the invention features a method fordetermining the safety of a drug. The method includes contacting heartvalve cells with the drug, and determining if the drug inducedproliferation of the cells, where the induction of proliferationindicates that the drug promotes heart valve degeneration.

[0018] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

[0019] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

[0020]FIG. 1 is a bar graph plotting the counts per minute (cpm) forvalvular cells treated with serum-free media (negative control), mediawith 10% fetal bovine serum (positive control), LDL alone, LDL pluspravastatin, or pravastatin alone.

[0021]FIG. 2 is a bar graph plotting the counts per minute (cpm) forAdβgal or AdeNOS infected valvular cells treated with conditioned media.Uninfected valvular cells treated with serum-free media were used as anegative control, while uninfected valvular cells treated mediacontaining 10% fetal bovine serum was used as a positive control.

DETAILED DESCRIPTION

[0022] The invention provides methods and materials related to heartvalves and the treatment of valvular heart disease. Specifically, theinvention provides non-murine heart valve cells and heart valve cusps aswell as methods for making heart valves. The invention also providesmethods and materials for (1) slowing heart valve degeneration,thrombosis, and calcification, (2) treating carcinoid heart disease, (3)identifying inhibitors of heart valve degeneration, thrombosis, andcalcification, and (4) determining the safety of drugs.

[0023] 1. Heart Valve Cells

[0024] A non-murine heart valve cell of the invention contains exogenousnucleic acid that encodes a polypeptide having nitric oxide synthaseactivity. Such heart valve cells can be used to make bioprosthetic heartvalves with significant durability since these cells produce a favorableextracellular matrix. Briefly, an extracellular matrix is any materialproduced by cells and secreted into the surrounding medium (e.g., thenoncellular portion of animal tissue). In broad terms, there are threemajor components of an extracellular matrix: fibrous elements (e.g.,collagen, elastin, and reticulin), link polypeptides (e.g., fibronectinand laminin), and space filling molecules (e.g., glycosaminoglycans).Although an extracellular matrix is produced by cells, it candramatically influence the behavior of tissue cells. In short, theproperties of an extracellular matrix have a large influence on theproperties of the tissue. Thus, the molecular makeup of a heart valve'sextracellular matrix is an important component determining thrombosis aswell as heart valve degeneration and calcification.

[0025] As described herein, non-murine heart valve cells containingexogenous nucleic acid that encodes a polypeptide having nitric oxidesynthase activity produce a different set of extracellular matrixmolecules than those produced by similar cells lacking exogenous nucleicacid that encodes a polypeptide having nitric oxide synthase activity.For example, unmodified heart valve cells exposed to harmful stimulisuch as hyperlipidemia produce fibronectin and osteopontin, while heartvalve cells of the invention produce little, if any, fibronectin orosteopontin in response to hyperlipidemia. Thus, any non-murine heartvalve cell can be given exogenous nucleic acid that encodes apolypeptide having nitric oxide synthase activity to change themolecular makeup of the extracellular matrix produced by a heart valvecell.

[0026] In addition, a heart valve cell within the scope of the inventioncan be used to make bioprosthetic heart valves with significantdurability since such cells do not proliferate excessively in responseto harmful stimuli such as hyperlipidemia. Briefly, hyperlipidemiarefers to an elevated concentration of lipids (e.g., cholesterol,triglycerides, and lipoproteins) in plasma. As described herein, heartvalve cells exposed to hyperlipidemic conditions exhibit excessiveproliferation and apoptosis that can lead to a malfunctioning heartvalve. However, non-murine heart valve cells containing exogenousnucleic acid encoding a polypeptide having nitric oxide synthaseactivity exhibit little, if any, proliferation or apoptosis in responseto harmful stimuli. Thus, any non-murine heart valve cell can be givenexogenous nucleic acid that encodes a polypeptide having nitric oxidesynthase activity to inhibit heart valve cell proliferation andapoptosis.

[0027] A non-murine heart valve cell is any non-murine cell obtainedfrom heart valve tissue or any non-murine cell that has been cultured insuch a way as to develop characteristics indistinguishable from those ofa cell obtained from heart valve tissue. For example, a non-murine heartvalve cell can be, without limitation, an endothelial cell or myocyteobtained from human heart valve tissue. In addition, a non-murine heartvalve cell can be, without limitation, a cell derived from a human stemcell that was cultured such that it resembles an endothelial cell ormyocyte obtained from human heart valve tissue. It is important to notethat a heart valve cell can be from any non-murine species having heartvalve tissue including, without limitation. mammals such as pigs, cows,horses, sheep, goats, monkeys, and humans. The term “murine” as usedherein refers to all species grouped within the Murinae taxon. Thus, allporcine cells would be considered non-murine cells. In addition, a heartvalve cell can be obtained from any heart valve including the tricuspid,pulmonary, mitral, and aortic valves of a non-murine mammal. It also isimportant to note that the heart valve cells of the invention can be inany physical state. For example, a heart valve cell of the invention canbe a cell within an organism's body, a cell maintained in tissueculture, or a cell that is or has been fixed, frozen, or otherwisetreated. Thus, a fixed non-murine heart valve cell containing nucleicacid encoding a polypeptide having nitric oxide synthase activity thatis stored at −70° C. is within the scope of the invention. When fixingcells, any fixative (e.g., glutaraldehyde under high pressure) can beused. When freezing cells, any temperature below freezing (e.g., 0, −20,and −70° C.) can be used. In addition, those skilled in the art willappreciate that media used to freeze cells will normally containingredients that help promote cell viability during freezing and thawingprocedures such as dimethyl sulfoxide.

[0028] The term “nucleic acid” as used herein encompasses both RNA andDNA, including cDNA, genomic DNA, and synthetic (e.g., chemicallysynthesized) DNA. The nucleic acid can be double-stranded orsingle-stranded. Where single-stranded, the nucleic acid can be thesense strand or the antisense strand. In addition, nucleic acid can becircular or linear.

[0029] The term “exogenous” as used herein with reference to nucleicacid and a particular cell refers to any nucleic acid that does notoriginate from that particular cell as found in nature. Thus, allnon-naturally occurring nucleic acid are considered to be exogenous to acell once introduced into the cell. It is important to note thatnon-naturally occurring nucleic acid can contain nucleic acid sequencesor fragments of nucleic acid sequences that are found in nature providedthe nucleic acid as a whole does not exist in nature. For example, anucleic acid containing a genomic DNA sequence within an expressionvector is considered to be a non-naturally-occurring nucleic acid, andthus is considered to be exogenous to a cell once introduced into thecell, since that nucleic acid as a whole (genomic DNA plus vector DNA)does not exist in nature. Thus, any vector, autonomously replicatingplasmid, or virus (e.g. retrovirus, adenovirus, or herpes virus) that asa whole does not exist in nature is considered to be anon-naturally-occurring nucleic acid. It follows that genomic DNAfragments produced by PCR or restriction endonuclease treatment as wellas cDNA's are considered to be a non-naturally-occurring nucleic acidsince they exist as separate molecules not found in nature. It alsofollows that any nucleic acid containing a promoter sequence andpolypeptide-encoding sequence (e.g., cDNA or genomic DNA) in anarrangement not found in nature is considered to be a non-naturallyoccurring nucleic acid.

[0030] It is also important to note that a nucleic acid that isnaturally occurring can be exogenous to a particular cell. For example,an entire chromosome isolated from a cell of person X would beconsidered an exogenous nucleic acid with respect to a cell of person Yonce that chromosome is introduced into Y's cell.

[0031] Any nucleic acid that encodes a polypeptide having nitric oxidesynthase activity (i.e., EC 1.14.13.39) can be used as described herein.Polypeptides having nitric oxide synthase activity include, withoutlimitation, nitric oxide synthase polypeptides designated as being type1, 2, or 3 (i.e., NOS1, NOS2, or NOS3). For example, a nucleic acidencoding NOS1, NOS2, or NOS3 can be given to a non-murine heart valvecell as described herein. In addition, a polypeptide having nitric oxidesynthase activity can be a polypeptide that is eithernaturally-occurring or non-naturally-occurring. A naturally-occurringpolypeptide is any polypeptide having an amino acid sequence as found innature, including wild-type and polymorphic polypeptides. Suchnaturally-occurring polypeptides can be obtained from any speciesincluding, without limitation, homo sapiens as well as other mammalianspecies such as murine, porcine, and bovine species and non-mammalianspecies such as insect (e.g., Drosophila and Anopheles), fish (e.g.,goldfish), and bird (e.g., chicken) species. For example, ratpolypeptides having nitric oxide synthase activity can have an aminoacid sequence as set forth in the GenBank® submissions with AccessionNumbers P29476, Q06518, Q62600, and AAC52782. Additional polypeptideshaving nitric oxide synthase activity can include, without limitation,bovine polypeptides (e.g., Accession Numbers P29473, 742990, 145946, andAAB22708), mouse polypeptides (e.g., Accession Numbers JN0609, P29477,and AAC52766), rabbit polypeptides (e.g., Accession Number AAB68663),guinea pig polypeptides (e.g., Accession Numbers AAD29751 and AAD29752),chicken polypeptides (e.g., Accession Number Q90703), dog polypeptides(e.g., Accession Number AAD39340), tobacco hornworm polypeptides (e.g.,Accession Number AAC61262), pig polypeptides (e.g., Accession NumbersAAB39539), and human polypeptides (e.g., Accession Numbers P29475,AAB60654, P35228, P29474, AAB49040, AAA36376, S28878, JX0345, CAA53950,BAA05652, and AAA36364).

[0032] A non-naturally-occurring polypeptide is any polypeptide havingan amino acid sequence that is not found in nature. Thus, anon-naturally-occurring polypeptide can be a mutated version of anaturally-occurring polypeptide, or an engineered polypeptide. Forexample, a non-naturally-occurring polypeptide having nitric oxidesynthase activity can be a mutated version of a naturally-occurringpolypeptide having nitric oxide synthase activity that retains at leastsome nitric oxide synthase activity. Possible mutations include, withoutlimitation, deletions, insertions, and base substitutions, as well ascombinations of deletions, insertions, and base substitutions. Anymethod including common molecular cloning techniques (e.g.,site-directed mutageneses) can be used to create a mutated version of anaturally-occurring polypeptide that retains at least some nitric oxidesynthase activity.

[0033] Nucleic acid encoding a polypeptide having nitric oxide synthaseactivity can be identified and obtained using any method. For example, anucleic acid encoding a nitric oxide synthase polypeptide having anamino acid sequence as set forth in GenBank® Accession Number CAA53950can be obtained using PCR. PCR refers to a procedure or technique inwhich target nucleic acid is amplified in a manner similar to thatdescribed in U.S. Pat. No. 4,683,195, and subsequent modifications ofthe procedure described therein. Generally, sequence information fromthe ends of the region of interest or beyond are used to designoligonucleotide primers that are identical or similar in sequence toopposite strands of a potential template to be amplified. Using PCR, anucleic acid sequence can be amplified from RNA or DNA. For example, anucleic acid sequence can be isolated by PCR amplification from totalcellular RNA, total genomic DNA, and cDNA as well as from bacteriophagesequences, plasmid sequences, viral sequences, and the like. When usingRNA as a source of template, reverse transcriptase can be used tosynthesize complimentary DNA strands.

[0034] In addition, nucleic acid and amino acid databases (e.g.,GenBank®) can be used to identify a nucleic acid sequence that encodes apolypeptide having nitric oxide synthase activity. Briefly, any nucleicacid or amino acid sequence having some homology (e.g., at least about30, 40, 50, 60, 70, 80, 90, 95, or 99% identity) to a known nitric oxidesynthase sequence can be used as a query to search GenBank®. Suchsearches can be performed over the Internet at, for example,www.ncbi.nlm.nih.gov.

[0035] Further, nucleic acid hybridization techniques can be used toidentify and obtain nucleic acid that encodes a polypeptide havingnitric oxide synthase activity. Briefly, any nucleic acid known toencode a polypeptide having nitric oxide synthase activity, or fragmentthereof, can be used as a probe to identify a similar nucleic acid byhybridization under conditions of low to high stringency. Such similarnucleic acid then can be isolated, sequenced, and analyzed to verifythat the encoded polypeptide exhibits nitric oxide synthase activity.

[0036] In general, high stringency conditions can be used to identifynucleic acid having a high degree of homology to a probe. Highstringency conditions can include the use of a denaturing agent such asform amide during hybridization, e.g., 50% formamide with 0.1% bovineserum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodiumphosphate buffer at pH 6.5 with 750 mM NaCl, and 75 mM sodium citrate at42° C. Another example is the use of 50% formamide, 5×SSC (0.75 M NaCl,0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.5), 0.1% sodiumpyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50μg/ml), 0.1% sodium lauryl sulfate (SDS), and 10% dextran sulfate at 42°C., with washes at 42° C. in 0.2×SSC and 0.1% SDS. Alternatively, lowionic strength and high temperature can be used for washing, forexample, 0.1×SSC (0.015 M NaCl/0.0015 M sodium citrate), 0.1% SDS at 65°C.

[0037] Moderate stringency conditions can be used to identify nucleicacid having a moderate degree of homology to a probe. Moderatestringency conditions can include the use of higher ionic strengthand/or lower temperatures for washing of the hybridization membrane,compared to the ionic strength and temperatures used for high stringencyhybridization. For example, a wash solution of 4×SSC (0.06 M NaCl/0.006M sodium citrate), 0.1% SDS can be used at 50° C., with a last wash in1×SSC at 65° C. Alternatively, a hybridization wash in 1×SSC at 37° C.can be used.

[0038] Low stringency conditions can be used to identify nucleic acidhaving a low degree of homology to a probe. Low stringency conditionscan include the use of higher ionic strength and/or lower temperaturesfor washing of the hybridization membrane, compared to the ionicstrength and temperatures used for moderate stringency hybridization.For example, a wash solution of 4×SSC (0.06 M NaCl/0.006 M sodiumcitrate), 0.1% SDS can be used at 37° C., with a last wash in 1×SSC at45° C. Alternatively, a hybridization wash in 2×SSC at 37° C. can beused.

[0039] Hybridization can be done by Southern or Northern analysis toidentify a DNA or RNA sequence, respectively, that hybridizes to aprobe. The probe can be labeled with a radioisotope such as ³²P, anenzyme, digoxygenin, or by biotinylation. The DNA or RNA to be analyzedcan be electrophoretically separated on an agarose or polyacrylamidegel, transferred to nitrocellulose, nylon, or other suitable membrane,and hybridized with the probe using standard techniques well known inthe art such as those described in sections 7.39-7.52 of Sambrook etal., (1989) Molecular Cloning, second edition, Cold Spring harborLaboratory, Plainview, N.Y. Typically, a probe is at least about 20nucleotides in length. For example, a probe corresponding to a 20nucleotide sequence within the GenBank® submission having AccessionNumber D26607 or M93718 can be used to identify a nucleic acid identicalor similar to the nucleic acid sequence encoding human endothelialnitric oxide synthase. In addition, probes longer or shorter than 20nucleotides can be used.

[0040] The exogenous nucleic acid contained within a cell of theinvention can be maintained within that cell in any form. For example,exogenous nucleic acid can be integrated into the genome of the cell ormaintained in an episomal state. In other words, a cell of the inventioncan be a stable or transient transformant.

[0041] In addition, any method can be used to introduce an exogenousnucleic acid into a cell. In fact, many methods for introducing nucleicacid into cells, whether in vivo or in vitro, are well known to thoseskilled in the art. For example, calcium phosphate precipitation,electroporation, particle bombardment, heat shock, lipofection,microinjection, and viral-mediated nucleic acid transfer are commonmethods for introducing nucleic acid into cells. Thus, well knownadenovirus-mediated nucleic acid transfer methods such as thosedescribed by Berkner et al. (Nucleic Acids Res., 11:6003-6020 (1983)),van Doren et al. (Mol. Cell. Biol., 4:1653-1656 (1984)), Ghosh-Choudhuryet al. (Biochem. Biophys. Res. Commun., 147:964-973 (1987)), McGrory etal. (Virology, 163:614-617 (1988)), and Gluzman et al. (In: EurkaryoticViral Vectors, Ed. Gluzman, Y. pages 187-192, Cold Spring HarborLaboratory (1982)) can be used to introduce exogenous nucleic acidencoding a polypeptide having nitric oxide synthase activity into aheart valve cell. In addition, naked DNA can be delivered directly tocells in vivo as describe elsewhere (U.S. Pat. Nos. 5,580,859 and5,589,466 including continuations thereof). Further, nucleic acid can beintroduced into cells by generating transgenic animals such astransgenic pigs, goats, sheep, cows, horses, dogs, cats, rabbits,baboon, monkeys, and chimpanzees. Several techniques known in the artcan be used to introduce nucleic acid into animals to produce thefounder lines of transgenic animals. Such techniques include, but arenot limited to, pronuclear microinjection (U.S. Pat. No. 4,873,191);retrovirus mediated gene transfer into germ lines (Van der Putten etal., Proc. Natl. Acad. Sci., USA, 82:6148-6152 (1985)); genetransfection into embryonic stem cells (Gossler A et al., Proc Natl AcadSci USA 83:9065-9069 (1986)); gene targeting into embryonic stem cells(Thompson et al., Cell, 56:313-321 (1989)); nuclear transfer of somaticnuclei (Schnieke A E et al., Science 278:2130-2133 (1997) and Wilmut Iet al., Nature 385:810-13 (1997)); and electroporation of embryos.

[0042] For a review of techniques that can be used to generate andassess transgenic animals, skilled artisans can consult Gordon (Intl.Rev. Cytol., 115:171-229 (1989)), and can obtain additional guidancefrom, for example: Hogan et al., “Manipulating the Mouse Embryo” ColdSpring Harbor Press. Cold Spring Harbor, N.Y. (1986); Krimpenfort etal., Bio/Technology, 9:844-847 (1991); Palmiter et al., Cell, 41:343-345(1985); Kraemer et al., “Genetic Manipulation of the Early MammalianEmbryo” Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1985);Hammer et al., Nature, 315:680-683 (1985); Purscel et al., Science,244:1281-1288 (1986); Wagner et al., U.S. Pat. No. 5,175,385; andKrimpenfort et al., U.S. Pat. No. 5,175,384.

[0043] Methods of identifying cells that contain exogenous nucleic acidalso are well known to those skilled in the art. Such methods include,without limitation, PCR and nucleic acid hybridization techniques suchas Northern and Southern analysis. For example, a nucleic acid fragmentfrom a known nitric oxide synthase nucleic acid sequence can be used asa probe in a Southern blot analysis to determine if a particular cellcontains nucleic acid corresponding to that probe.

[0044] In addition to containing an exogenous nucleic acid that encodesa polypeptide having nitric oxide synthase activity, a cell of theinvention can express the encoded polypeptide having nitric oxidesynthase activity. Any method can be used to express a polypeptide froman exogenous nucleic acid. For example, a nucleic acid can beconstructed such that a regulatory element promotes the expression of anucleic acid sequence that encodes a polypeptide. Typically, regulatoryelements are DNA sequences that regulate the expression of other DNAsequences at the level of transcription. Thus, regulatory elementsinclude, without limitation, promoters, enhancers, and the like.

[0045] Many methods can be used to identify cells that contain exogenousnucleic acid and express the encoded polypeptide. Such methods include,without limitation, PCR, RT-PCR, nucleic acid hybridization techniques,immunohistochemistry, and biochemical techniques. Briefly, RT-PCRtechniques and Northern analysis can be used to assess the expression ofnitric oxide synthase mRNA, while immunocytochemistry using anti-nitricoxide synthase antibodies can be used to assess the expression of nitricoxide synthase polypeptide. For example, detection of nitric oxidesynthase-immunoreactivity after introduction of an exogenous nucleicacid that encodes a polypeptide having nitric oxide synthase activityinto a cell that does not normally express a nitric oxide synthasepolypeptide can indicate that cell not only contains the introducedexogenous nucleic acid but also expresses the encoded nitric oxidesynthase polypeptide from that introduced exogenous nucleic acid.Likewise, biochemical detection of nitric oxide synthase activity withina cell can indicate that particular cell expresses the encoded nitricoxide synthase polypeptide.

[0046] 2. Heart Valve Cusps

[0047] The invention also provides heart valve cusps that contain a cell(e.g., a non-murine cell) with exogenous nucleic acid encoding apolypeptide having nitric oxide synthase activity. Such heart valvecusps can be used to make heart valves (e.g., bioprosthetic heartvalves) that can replace native heart valves within mammals (e.g.,humans). It is important to note that the heart valve cusps of theinvention can be used to make any type of heart valve including valveshaving two or three cusps (e.g., bicuspid and tricuspid heart valves).In addition, heart valve cusps can be obtained from any non-murinespecies including, without limitation, mammals such as pigs, cows,horses, sheep, goats, monkeys, and humans. Thus, a heart valve cusp thatis used to replace a native cusp within a mammal can be an autograft,allograft, or xenograft. The term “autograft” refers to a surgical graftof tissue taken from one part of the body and placed in another site ofthe same individual. The term “allograft” refers to a surgical graft oftissue taken from one individual's body and placed into anotherindividual's body with the two individuals being allogeneic at one ormore loci such as a histocompatibility loci. The term “xenograft” refersto a surgical graft of tissue taken from one species and placed into anindividual of a different species.

[0048] A heart valve cusp of the invention can be an isolated heartvalve cusp. The term “isolated” as used herein with reference to a heartvalve cusp refers to any heart valve cusp that has been removed from anorganism's heart. For example, a heart valve cusp that has beensurgically removed from a pig's heart is an isolated heart valve cusp.It will be understood that a heart valve cusp that was removed from apig's heart and placed within a human patient's heart is an isolatedheart valve cusp since that cusp has been removed from a pig's heart.

[0049] It is noted that a heart valve cusp of the invention can be acusp that is not removed from an organism's body. In other words,exogenous nucleic acid encoding a polypeptide having nitric oxidesynthase activity can be introduced into an organism's heart valve cuspin vivo or in vitro. For example, a human patient can be given anadenoviral vector such that a cell of a heart valve cusp acquiresnucleic acid that encodes a polypeptide having nitric oxide synthaseactivity. In this case, the expression to the polypeptide having nitricoxide synthase activity can lead to significant heart valve durability.In addition, the human patient can be given an inhibitor ofhydroxymethylglutaryl coA reductase activity (i.e., EC 1.1.1.34) such aspravastatin, atorvastatin, simvastatin, or lovastatin. As describedherein, administration of an inhibitor of hydroxymethylglutaryl coAreductase activity can reduce heart valve calcification and degenerationwithin a mammal.

[0050] Like the cells of the invention, a heart valve cusp can be in anyphysical state. For example, a heart valve cusp of the invention can bewithin an organism's body, maintained in tissue culture, or fixed,frozen, or otherwise treated. Thus, heart valve cusps that have beenremoved from an organism's body as well as those within an organism'sbody are within the scope of the invention provided they contain anon-murine cell having exogenous nucleic acid that encodes a polypeptidehaving nitric oxide synthase activity.

[0051] It is noted that any number of the cells constituting a heartvalve cusp from a single cell to all the cells can contain exogenousnucleic acid encoding a polypeptide having nitric oxide synthaseactivity. For example, about one, two, five, ten, or fifty percent ofthe cells of a heart valve cusp can contain exogenous nucleic acidencoding a polypeptide having nitric oxide synthase activity.

[0052] 3. Use of Hydroxymethylglutaryl coA Reductase Inhibitors

[0053] The invention also provides methods for slowing the onset, orrate of progression, of heart valve degeneration, thrombosis, andcalcification. In addition, the provided methods can reduce a mammal'ssusceptibility to, or prolong a mammal's resistance against, heart valvedegeneration, thrombosis, and calcification. These methods include (a)identifying a mammal at risk of developing heart valve degeneration,thrombosis, or calcification, and (b) administering an inhibitor ofhydroxymethylglutaryl coA reductase activity. Mammals at risk ofdeveloping heart valve degeneration, thrombosis, or calcificationinclude, without limitation, mammals that have received heart valvereplacement surgery as well as those mammals having congenital valvulardisease (e.g., bicuspid valvular disease). Such mammals can beidentified using standard clinical diagnostic techniques (e.g.,echocardiography and cardiac catheterization). In addition, medicalhistory records can be used to identify patients at risk of developingheart valve degeneration, thrombosis, or calcification. For example,medical history records can be used to identify patients having hadheart valve replacement surgery while between the age of 30 and 50 yearssince such patients are at risk of developing heart valve degeneration,thrombosis, or calcification.

[0054] Once a mammal in need of treatment is identified, an inhibitor ofhydroxymethylglutaryl coA reductase activity can be administered.Although not limited to any particular mode of action, administration ofan inhibitor of hydroxymethylglutaryl coA reductase activity presumablycauses an increase in nitric oxide synthase activity within valvulartissue. As described herein, an increase in nitric oxide synthaseactivity within valvular tissue can alter the molecular makeup of theheart valve's extracellular matrix as well as inhibit heart valve cellproliferation and apoptosis normally induced by harmful stimuli.Inhibitors of hydroxymethylglutaryl coA reductase activity include,without limitation, statin drugs such as pravastatin, atorvastatin,simvastatin, and lovastatin.

[0055] Any method can be used to determine if hydroxymethylglutaryl coAreductase activity was inhibited. For example, the level of expressionof a particular nitric oxide synthase enzyme can be determined bymeasuring mRNA or polypeptide levels within a tissue sample. Moreover,clinical methods that can assess the degree of hydroxymethylglutaryl coAreductase or nitric oxide synthase activity can be used to determinewhether hydroxymethylglutaryl coA reductase activity was inhibited.

[0056] Typically, a pharmaceutically effective amount of an inhibitor ofhydroxymethylglutaryl coA reductase activity is administered to a mammalin need of treatment. A pharmaceutically effective amount of aninhibitor of hydroxymethylglutaryl coA reductase activity refers to anyamount that does not cause significant toxicity to the host and inhibitshydroxymethylglutaryl coA reductase activity. Such an amount can bedetermined by assessing the clinical symptoms of a patient before andafter administering a fixed amount of a particular material. Inaddition, the pharmaceutically effective amount of a particular materialadministered to a host can be adjusted according to the host's responseand desired outcomes. Significant toxicity can vary for each particularpatient and depends on multiple factors including, without limitation,the patient's age, tolerance to pain, and disease state.

[0057] In addition, any of the materials described herein can beadministered to any part of the host's body including, withoutlimitation, the blood stream, lungs, intestines, and the like. Thus, aninhibitor of hydroxymethylglutaryl coA reductase activity can beadministered by intravenous injection, inhalation, or oraladministration. For example, an aerosol preparation containing aninhibitor of hydroxymethylglutaryl coA reductase activity can be givento a patient by inhalation. It is noted that the duration of treatmentwith any of the materials described herein can be any length of timefrom as short as one day to as long as a lifetime (e.g., many years).For example, an inhibitor of hydroxymethylglutaryl coA reductaseactivity can be administered at some frequency over a period of tenyears. It is also noted that the frequency of treatment can be variable.For example, an inhibitor of hydroxymethylglutaryl coA reductaseactivity can be administered once (or twice, three times, etc.) daily,weekly, monthly, or yearly.

[0058] Preparations for administration can include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents include, without limitation, propylene glycol,polyethylene glycol, vegetable oils, and injectable organic esters.Aqueous carriers include, without limitation, water as well as alcohol,saline, and buffered solutions. Preservatives, flavorings, and otheradditives such as, for example, antimicrobials, anti-oxidants, chelatingagents, inert gases, and the like may also be present.

[0059] 4. Treating Carcinoid Heart Disease

[0060] Carcinoid heart disease refers to a cardiac manifestation ofmalignant carcinoid syndrome. Briefly, carcinoid heart disease is aunique form of fibrosis involving primarily the endocardium of the rightheart. In general, fibrous deposits tend to cause constriction of thetricuspid and pulmonary heart valves. Mammals having carcinoid heartdisease can be identified using common medical diagnostic techniques.For example, echocardiography can be used to identify human patientsthat have carcinoid heart disease. In addition, 5-hydroxyindole aceticacid (5-HIAA) levels within urine can be measured to identify mammalshaving a carcinoid tumor. Typically, an elevated level of 5-HIAA in apatient's urine indicates the presence of a carcinoid tumor. It is notedthat the normal range of 5-HIAA within urine collected for a 24 hourtime period is about 0.0 mg to about 6.0 mg (i.e., about 0-6 mg 5-HIAAin urine per 24 hours).

[0061] As described herein, administration of a serotonin receptorinhibitor (e.g., a 5HT_(1B) receptor inhibitor) can reduceserotonin-induced proliferation of valvular tissue. Briefly, serotoninwas found to induce proliferation of heart valve cells in a 5HT_(1B)selective manner, indicating the involvement of 5TH_(1B) receptoractivity in the proliferation process. Thus, carcinoid heart disease canbe treated by administering a serotonin receptor inhibitor. Suchinhibitors include, without limitation, β-blockers such as pindolol,meththiotepin, metoprolol, and paldolol.

[0062] Typically, a pharmaceutically effective amount of a serotoninreceptor inhibitor is administered to a mammal in need of treatment. Apharmaceutically effective amount of a serotonin receptor inhibitorrefers to any amount that does not cause significant toxicity to thehost and inhibits serotonin receptor activity. Such an amount can bedetermined by assessing the clinical symptoms of a patient before andafter administering a fixed amount of a particular material. Inaddition, the pharmaceutically effective amount of a particular materialadministered to a host can be adjusted according to the host's responseand desired outcomes. Significant toxicity can vary for each particularpatient and depends on multiple factors including, without limitation,the patient's age, tolerance to pain, and disease state.

[0063] 5. Identifying Inhibitors of Heart Valve Degeneration andDetermining Drug Safety

[0064] The invention also provides methods and materials for identifyingcompounds that can be used to inhibit heart valve degeneration,thrombosis, and calcification. Such methods include (a) contacting heartvalve cells with a stimulant to promote proliferation, (b) contactingthe proliferating cells with a test compound, and (c) determining if thetest compound reduced cell proliferation. Any type of stimulant can beused provided heart valve cell proliferation is promoted. Suchstimulants can include, without limitation, growth factors such as basicfibroblast growth factor, PDGF, and TGF-β as well as solutions thatcreate hyperlipidemic conditions. In addition, any type of compound canbe used as a test compound including, without limitation, polypeptides,nucleic acids, carbohydrates, lipids, synthetic chemicals, inorganiccompounds, and any other compound such as those listed in the MerckIndex (12th Edition, March 1996, available from the Merck PublishingGroup, Rahway, N.J.). A test compound also can be a member of acombinatorial library. Compounds identified using the methods of theinvention can be further evaluated, detected, cloned, sequenced, and thelike.

[0065] Any method can be used to measure proliferation. For example,immunocytochemistry can be used to monitor proliferation markers such asproliferating cell nuclear antigen (PCNA).

[0066] In addition, biological assays designed to detect proliferationby measuring incorporation of radio-labeled nucleotides (e.g., tritiatedthymidine) can be used. Thus, a compound that inhibits heart valvedegeneration can be identified by monitoring the proliferation ofporcine heart valve cells stimulated with a growth factor before andafter treatment with a test compound.

[0067] In another embodiment, apoptosis instead of proliferation can beused to identify therapeutic compounds. For example, a method within thescope of the invention can include (a) contacting heart valve cells witha stimulant to promote apoptosis, (b) contacting the treated cells witha test compound, and (c) determining if the test compound reducedapoptosis. Any type of stimulant can be used provided heart valve cellapoptosis is promoted. Such stimulants can include, without limitation,serotonin. fenfluramine, and cholesterol. In addition any method can beused to measure apoptosis. For example, immunocytochemistry can be usedto monitor apoptosis markers such as CPP32. In addition, a TdT-mediateddUTP-biotin nick end-labeling technique (TUNEL) can be used to measureapoptosis. Thus, a compound that inhibits heart valve degeneration canbe identified by monitoring the apoptosis of porcine heart valve cellsstimulated with an apoptosis-inducing factor before and after treatmentwith a test compound.

[0068] In addition, the methods and materials of the invention can beused to assess the safety of any substance given to a mammal. Forexample, any new drug designed for human use can be tested for adverseaffects on heart valve tissue. In general, the substance to be tested isapplied to heart valve cells after which the heart valve cells areexamined for any increase in proliferation and/or apoptosis, or anychange in the makeup of the extracellular matrix. Any increase in heartvalve cell proliferation and/or apoptosis can indicate that thesubstance promotes heart valve degeneration.

[0069] The invention will be further described in the followingexamples, which do not limit the scope of the invention described in theclaims.

EXAMPLES Example 1 Effects of Hyperlipidemia on Heart Valve Tissue

[0070] Rabbits were used since they have a propensity to developendothelial dysfunction when subjected to a cholesterol diet. Theeffects of feeding New Zealand White Rabbits a diet supplemented with 1%cholesterol (wt/wt; Purina Mills, Woodmont, Ind.) were studied. Briefly,the rabbits were fed cholesterol for ten weeks, while control rabbitsreceived a normal diet. After ten weeks, the rabbits were anesthetizedusing intramuscular ketamine (68 mg/kg), xylazine (9 mg/kg), andaceprmazine (2.3 mg/kg). Heparin (100 U/kg) was administeredintracardially, and the rabbits were killed four to five minutes laterwith an overdose of a pentobarbital sodium solution. The thorax wasopened widely, and the heart removed for histologic, RT-PCR, andimmunostaining analysis of the aortic heart valve. After removal, theaortic heart valves were either frozen in liquid nitrogen, or fixed in10% formalin and embedded in parafilm. The valvular appearancehistology, and immunostaining for fibronectin were examined to determinethe effects of hyperlipidemia.

[0071] Aortic heart valves from the hyperlipidemic rabbits exhibited athickening of the valve cusp with accompanying lipid accumulation. Theaortic heart valves from control rabbits did not exhibit cusp thickeningor lipid accumulation. Histological evaluation of the aortic valves fromhyperlipidemic rabbits revealed a definite fibroplastic proliferation ofthe subendothelial layer as well as a marked accumulation of lipid inwhat appeared to be foam cells. Similar lesions were found in theprogression of atherosclerosis to advanced lesions within arteries.Aortic heart valves from control rabbits did not exhibit fibroplasticproliferation or lipid accumulation.

[0072] Fibronectin is a dimeric glycoprotein found in the extracellularmatrix of most tissues. Immunostaining for fibronectin in aortic heartvalves from hyperlipidemic rabbits revealed marked staining for thisglycoprotein, while aortic heart valves from control rabbits exhibitedvery little fibronectin immunoreactivity. In addition, the fibronectinstaining in aortic heart valves from hyperlipidemic rabbits was detecteddiffusely in the endothelial and subendothelial layer. Moreover, usingRT-PCR, eNOS mRNA was detected in aortic heart valve tissue from boththe hyperlipidemic and control rabbits. Taken together, these resultsindicate that a stimulus such as hyperlipidemia can lead to significantchanges in not only the proliferative state of heart valve cells butalso the molecular makeup of the extracellular matrix of heart valves.

[0073] In another experiment, osteopontin expression was evaluated.Osteopontin is a non-collagenous bone matrix protein found to be presentin the hypercholesterolemic aortic valves. New Zealand rabbits (n=8)were fed with a 1% cholesterol diet for 12 weeks. Eight control animalswere fed a normal chow diet. Aortic valves were cut and foam cells wereidentified by immunostaining for macrophage antibody. Cell proliferationwas assessed by immunostaining for proliferating nuclear antigen (PCNA),and osteopontin (OP) for calcification. Transmission electron microscopyimmunogold labeling confirmed osteopontin expression.

[0074] Atherosclerotic lesions were present in all cholesterol treatedrabbit valves as compared to controls. These lesions stain positive forfoam cell formation. There was increased osteopontin expression in theatherosclerotic lesion of the aortic valves and a thousand-fold increasein the number of proliferating cells as compared to the controls.Cholesterol levels in the rabbits: the cholesterol treated rabbits had acholesterol level of 1036+/−96 mg/dL, the normal rabbits had acholesterol level of 42.28+/−mg/dL. Osteopontin expression was confirmedby immunogold labeling in the hypercholesterolemic valves. Electronmicroscopy analysis revealed that immunogold label is present primarilyin matrix vesicles embedded in collagen fibrils which is characteristicof calcification. Further, these heavily labeled areas were enriched incholesterol deposition.

[0075] These results indicate that hypercholesterolemia inducesatherosclerotic lesions in rabbit aortic valves, as evidenced by foamcell infiltration and an increase in cellular proliferation. Inaddition, these results indicate that hypercholesterolemia stimulatesosteopontin production. Thus, hypercholesterolemia may stimulate thecalcification process present in calcific aortic valve disease.

Example 2 Effects of HMG CoA Reductase Inhibitors and Oxidative Stresson Heart Valve Tissue in Vitro

[0076] Valvular endothelial and subendothelial tissues were obtainedfrom mature pigs and used to create primary explant cultures. Briefly,the cells were isolated from the cardiac aortic valves by collagenasedigestion, and cultured in medium 199 with 10% (v/v) heat-inactivatedfetal bovine serum at 37° C. in a humidified atmosphere of 5% CO₂ inair. For each experiment, the cells were used between the 3rd and 7thpassage. Cells were grown to about 80 percent confluence in 24-wellplates. Once at about 80 percent confluence, the cells were incubated inserum-free medium for 24 hours to arrest growth. After the 24 hourincubation, LDL (10 μg/mL) alone, pravastatin (100 nM) alone, or LDL (10μg/mL) plus pravastatin (100 nM) was added to the wells, and the cellsincubated for 18 hours. Controls were cells treated with eitherserum-free media or media containing 10% fetal bovine serum. Pravastatinis an HMG CoA reductase inhibitor obtained from Bristol Meyer Squibb.After the 18 hour incubation, the cells were incubated in the presenceof tritiated thymidine (1 μCi/well) for four hours and a thymidineuptake assay performed to assess cellular proliferation according to thestandard techniques described by Johnson et al. (J. Mol Cell. Card.19:1185-93 (1987)). Newly synthesized DNA was identified byincorporation of radioactivity into acid-precipitated cellular material.All samples were assayed in quadruplicate wells.

[0077] Treatment of the valvular cells with LDL alone resulted insubstantial proliferation as compared to cells incubated in serum-freemedia (FIG. 1). In fact, treatment with LDL alone resulted in moreproliferation than that observed for cell incubated in media containing10% fetal bovine serum (positive control). In addition, treatment withLDL plus Pravastatin resulted in substantially less proliferation thanthat observed for cells treated with LDL alone. Thus, valvular cellsproliferate in response to LDL, and this proliferation can be inhibitedby Pravastatin.

[0078] In another experiment, cells were grown to about 80 percentconfluence and then incubated in serum-free medium for 24 hours toarrest growth. After the 24 hour incubation, the cells were incubated inserum-free media containing 100 nM pravastatin for 24 hours. Controlcells were incubated in serum-free media only. After the 24 hourincubation, the cultured cells were collected and homogenized in a lysisbuffer (50 mM Tris-HCl, 1% NP-40, 1 mM EGTA, 1% mercaptoethanol, 100 mMleupeptin, and 1 mM PMSF). After pelleting nuclei, the supernatant wasboiled in Laemmli loading buffer and separated by SDS-PAGE (7.5%acrylamide gel). Once separated, the polypeptides were electroblottedonto nitrocellulose and probed with a monoclonal antibody to endothelialnitric oxide synthase (eNOS mAb; Transduction Laboratory; Lexington,Ky.). To ensure efficient polypeptide transfer, gels were stained withCoomasie Blue.

[0079] A Western blot analysis revealed that valvular cells containsubstantially more eNOS polypeptide after pravastatin treatment thanthat observed for cells treated with serum-free media alone. Theseresults indicate HMG CoA reductase inhibitors can induce eNOSpolypeptide expression within valvular cells.

[0080] To assess nitric oxide synthase activity, the aortic valveendothelial cell lysates were evaluated using an NADPH disphorasereaction. Briefly, each lysate was incubated for 30 minutes in a NOSassay buffer containing ³H-L-arginine, NADPH (100 mM),tetrahydrobiopterin (1 mM), calmodulin (10 mM), and CaCl₂ (25 mM). Thereaction was quenched with ice cold stop buffer (2 mM EDTA, 20 mM Hepes,pH 5.5). The mixture was passed over a Dowex AG50WX-8 Na form resin andwashed with one mL stop buffer. Nitric oxide synthase activity wascalculated to be the amount of L-arginine converted to L-citrulline thatwas inhibited by 1-NAME. A scintillation counter was used to determinethe amount of radioactivity.

[0081] Valvular cells treated with pravastatin exhibited about 100-foldmore nitric oxide synthase activity than that observed in cells treatedwith serum-free media alone. Thus, HMG CoA reductase inhibitors canincrease nitric oxide synthase activity in valvular cells.

Example 3 Adenoviral Vectors

[0082] Adenoviral vectors containing a cDNA encoding eitherβ-galactosidase or eNOS were constructed using methods similar to thosedescribed by Tsutsui et al. (Arterio. Thromb. Vascul. Biol. 18:1231-1241(1998). Briefly, each vector was produced by cloning the cDNA ofinterest into a plasmid containing a subsegment of the adenoviralgenome. Each resulting construct then was cotransfected into humanE1a-293 cells along with adenoviral DNA containing an E1a deletion.Adenoviruses containing a E1a deletion are replication defective,however, viral replication can occur within E1a-293 cells since thesecells provide E1a products in trans. Through homologous recombination,recombinant adenoviral vectors containing the cDNA of interest wereproduced within the 293 cells. The recombinant adenoviral vectors wereisolated and propagated. Once collected, each adenoviral vector stockwas stored at −70° C. in 0.01M Tris, 0.01M MgCl, and 10% glycerol. Therecombinant adenoviral vector containing the β-galactosidase cDNA wasdesignated Adβgal, while the eNOS containing adenoviral vector wasdesignated AdeNOS.

Example 4 Effects of eNOS Expression on Valvular Cell Proliferation

[0083] Valvular endothelial and subendothelial tissues were obtainedfrom mature pigs and used to create primary explant cultures. Briefly,the cells were isolated from the cardiac aortic valves by collagenasedigestion, and cultured in medium 199 with 10% (v/v) heat-inactivatedfetal bovine serum at 37° C. in a humidified atmosphere of 5% CO₂ inair. The cells were used between the 3rd and 7th passage. For eachexperiment, the cells were crown to about 80 percent confluence in24-well plates. Once at about 80 percent confluence, the cells wereincubated in serum-free medium for 24 hours to arrest growth. After the24 hour incubation, the cells were infected with either Adβgal orAdeNOS. Briefly, virus particles (200 MOI) were incubated with the cellsfor one hour. After the incubation, the cells were wash two times withPBS and cultured with serum-free media for 24 hours to allow expressionof either β-gal or eNOS. After culturing, the cells were incubated withconditioned media for 18 hours. Control cells were incubated in eitherserum-free media (negative control) or media containing 10 percent fetalbovine serum (positive control). After the 18 hour incubation, the cellswere incubated in the presence of tritiated thymidine (1 μCi/well) forfour hours and a thymidine uptake assay performed to assess cellularproliferation according to the standard techniques described by Johnsonet al. (J Mol. Cell. Card. 19:1185-93 (1987)). Newly synthesized DNA wasidentified by incorporation of radioactivity into acid-precipitatedcellular material. All samples were assayed in duplicate wells.

[0084] The conditioned media used to treat the cells was obtained fromprimary porcine valve explant cultures as follows. Cells were grown toconfluence and then incubated for 24 hours in serum-free media. Afterthe 24 hour incubation, 10 μg/mL of LDL was added and the culturesincubated for another 24 hours. After this second 24 hour incubation,the supernatant was collected and the cells discarded. This supernatantwas used as conditioned media.

[0085] Valvular cells infected with Adβgal and treated with conditionedmedia exhibited much more proliferation than that observed fromsimilarly treated valvular cells infected with AdeNOS (FIG. 2). Thus,eNOS expression can reduce the proliferative response of valvular cellsnormally induced by oxidative conditions.

Example 5 Analysis of Bioprosthetic Heart Valves

[0086] Heart valve tissue is collected from porcine subjects obtainedfrom a slaughterhouse. For human valvular tissue, a cadaver is used.Optionally, the ROSS procedure is used to remove a patient's pulmonicvalve and substitute it for their aortic valve. Prior to using the humanpulmonic valve, however, it is treated as described herein.

[0087] Once collected, the porcine or human heart valve tissue is storedin a cardioplegic solution oxygenated with carbogen (95% O₂, 5% CO₂).The cardioplegic solution contains 152 mM Na⁺, 3.6 mM K⁺, 135 mM Cl⁻, 25mM HCO₃ ⁻, 0.6 mM Mg²⁺, 1.3 mM H₂PO₄ ⁻, 0.6 mM SO₄ ²⁻, 2.5 mM Ca²⁺, 11.2mM glucose, 30 mM 2,3 butanedione-monoxime, and 10 IU/L insulin. Thecardioplegic solution can protect the myocardium while outside a body.An adenoviral nucleic acid delivery system is used to deliver cDNAsencoding β-galactosidase and/or eNOS to the valvular endothelial cellsof the heart valve tissue in vitro such that nitric oxide production canbe assessed within the cells.

[0088] AdeNOS is used to infect human and porcine aortic valves. TheAdβgal is used as a control to assess the degree of infection. Onceinfected, each tissue optionally is examined to determine the level ofNOS activity using an NADPH disphorase reaction in cells. In addition,the infected tissue optionally is tested for nitrite production.Briefly, the tissue is incubated for two hours in a Krebs solutioncontaining L-arginine (100 AM) and the calcium ionophore A23 187 (1 μM;Sigma catalog number C7522) After incubation, the solution is collectedand analyzed for nitrite concentration using a spectrofluorometricassay. Further, fibronectin and osteopontin production by each tissueoptionally is examined using immunocytochemistry.

[0089] In another set of experiments, heart valve tissue is infectedwith the AdeNOS, incubated for 24-36 hours, and frozen at −70° C.Presumably, infecting the heart valve tissue with AdeNOS prior tofreezing stabilizes the endothelium and extracellular matrix in additionto decreasing the amount of injury procured by the freezing process.Once thawed, the tissue is surgically implanted into a sheep or humanpatient. At various time points after implantation, the heart valvetissue is analyzed for degeneration, thrombosis, and calcification.

[0090] In yet another set of experiments, heart valve tissue is infectedwith the AdeNOS, incubated for 24-36 hours, and fixed. Presumably,infecting the heart valve tissue with AdeNOS prior to fixationstabilizes the endothelium and extracellular matrix in addition todecreasing the amount of injury procured by the fixation process. Oncefixed, the tissue is surgically implanted into a sheep or human patient.At various time points after implantation, the heart valve tissue isanalyzed for degeneration, thrombosis, and calcification.

Example 6 Frozen Bioprosthetic Heart Valves

[0091] Heart valve tissue is collected from porcine subjects obtainedfrom a slaughterhouse. For human valvular tissue, a cadaver is used.Optionally, the ROSS procedure is used to remove a patient's pulmonicvalve and substitute it for their aortic valve. Prior to using the humanpulmonic valve, however, it is treated as described herein.

[0092] Once collected, the porcine or human heart valve tissue is storedin a cardioplegic solution oxygenated with carbogen (95% O₂, 5% CO₂).The cardioplegic solution contains 152 mM Na⁺, 3.6 mM K⁺, 135 mM Cl⁻, 25mM HCO₃ ⁻, 0.6 mM Mg²⁺, 1.3 mM H₂PO₄ ⁻, 0.6 mM SO₄ ²⁻, 2.5 mM Ca²⁺, 11.2mM glucose, 30 mM 2,3 butanedione-monoxime, and 10 IU/L insulin. Thecardioplegic solution can protect the myocardium while outside a body.This tissue then is infected with an adenoviral vector. Briefly, anadenoviral nucleic acid delivery system is used to deliver a cDNAencoding eNOS to the valvular endothelial cells of the heart valvetissue such that nitric oxide synthase activity is increased within thecells. Once infected, the heart valve tissue is incubated for about 12to about 36 hours (e.g., about 24-36 hours), and frozen at+70° C. Thisfrozen heart valve tissue is thawed and implanted within a mammaliansubject requiring heart valve replacement.

Example 7 Fixed Bioprosthetic Heart Valves

[0093] Heart valve tissue is collected from porcine subjects obtainedfrom a slaughterhouse. For human valvular tissue, a cadaver is used.Optionally, the ROSS procedure is used to remove a patient's pulmonicvalve and substitute it for their aortic valve. Prior to using the humanpulmonic valve, however, it is treated as described herein.

[0094] Once collected, the porcine or human heart valve tissue is storedin a cardiopiegic solution oxygenated with carbogen (95% O₂, 5% CO₂).The cardioplegic solution contains 152 mM Na⁺, 3.6 mM K⁻, 135 mM Cl⁻, 25mM HCO₃ ⁻, 0.6 mM Mg²⁺, 1.3 mM H₂PO₄ ⁻, 0.6 mM SO₄ ²⁻, 2.5 mM Ca²⁺, 11.2mN glucose, 30 mM 2,3 butanedione-monoxime, and 10 IU/L insulin. Thecardioplegic solution can protect the myocardium while outside a body.This tissue then is infected with an adenoviral vector. Breifly, anadenoviral nucleic acid delivery system is used to deliver a cDNAencoding eNOS to the valvular endothelial cells of the heart valvetissue such that nitric oxide synthase activity is increased within thecells. Once infected, the heart valve tissue is incubated for about 12to about 36 hours (e.g., about 24-36 hours), and fixed. This fixed heartvalve tissue is implanted within a mammalian subject requiring heartvalve replacement.

Example 8 Carcinoid Heart Disease

[0095] Heart valve tissue was obtained from patients with and withoutcarcinoid heart disease. Patients with carcinoid heart disease wereidentified by retrospective review of a tissue registry. In addition,patients were identified as having carcinoid heart disease by measuring5-HIAA levels within urine as well as by histologically examining heartvalve tissue for a normal endothelial layer and a subendothelial layercontaining proliferating cells and an increased extracellular matrix.

[0096] Gross inspection of the heart valves from patients with carcinoidheart disease at the time of valve replacement reveal a smooth appearingendocardial surface. In addition, these heart valves containedplaque-like lesions consisting of a thickened subendothelial cell layer.In fact, these subendothelial cells compose the majority of theendocardial plaque surface. Conversely, normal control heart valvesexhibited a clear, glistening valvular cusp with no visible evidence ofendocardial plaque lesions. The valve surface was intact.

[0097] To examine collagen synthesis, the heart valves were stained withMasson trichrome. A marked increase in the collagen identified by theMasson trichrome stain was observed in the carcinoid valves as comparedto the normal valves. In addition, younger lesions exhibited less blueintensity than older lesions indicating that younger lesions have alesser amount of collagen synthesis than the older lesions.

[0098] PCNA immunostaining was performed to examine in situproliferation ex vivo. Briefly, slides containing the heart valve tissuewere deparaffinized and rehydrated through the following solutions:xylene twice for five minutes, 100% ethanol twice for ten seconds, and95% ethanol twice for ten seconds. Endogeneous peroxidase activity wasblocked by incubating the slide in a 50% volume H₂O/50% volume methanolsolution at room temperature for ten minutes followed by a rinsing withrunning tap water. Non-specific polypeptide binding sites were blockedby applying 5% normal horse serum diluted in PBS/0.05% Tween 20 (pH7.2-7.4; obtained from Pierce Chemical Co.) to each slide for tenminutes at room temperature. The serum was blotted off, and primaryantibody diluted in 1% normal horse serum plus PBS/0.05% Tween 20applied for an overnight incubation period in a humidity chamber at 4°C. The PCNA antibody (obtained from Dako; Carpinteria, Calif. was usedat a 1:7500 dilution. As described below, the heart valve tissue alsowas stained with an 5HT_(1B) antibody (1:75 dilution; obtained fromSanta Cruz Biotech; Santa Cruz, Calif.). After overnight incubation, theprimary antibody was rinsed off with tap water, the slides blotted, anda biotinylated secondary antisera cocktail containing goat anti-mouseIgG (diluted 1:400) added. After a 30 minute room temperatureincubation, the slides were rinsed with running tap water, and incubatedwith streptavidin-horseradish peroxidase diluted 1:500 in PBS/0.05%,Tween 20 with 1% normal goat serum for 30 minutes at room temperature.The slides then were rinsed with running tap water, and exposed to colordevelopment using 3-amino-9-ethylcarbazole as a substrate solution(Sigma) for 15 minutes at room temperature. The slides werecounterstained with hematoxylin for 30 seconds, and a coverslip applied.

[0099] PCNA quantitation was performed by automated digital imageanalysis. Immunoperoxidase stains treating the tissue were measured bycomparing intensity of light passing through the glass slide on whichthe cells reside. This light was captured by a video camera set at aspecific wavelength. The absorption value of each pixel represents thelevel of light absorbed by the cellular constituents in a finite area.These pixels were then quantified for the analysis of proliferatingcellular nuclei.

[0100] PCNA immunostaining of heart valves from carcinoid heart diseasepatients revealed a marked increase in the number of positive brownstaining nuclei as compared to normal control tricuspid valves,corresponding to an upregulation of DNA polymerase in the carcinoidvalves. It is noted that DNA polymerase is a marker of cellproliferation. Quantitation of the number of PCNA positive stainingnuclei in the carcinoid tricuspid valve versus the normal tricuspidvalve revealed a 35-fold increase in the number of positive nuclei ascompared to the control (p<0.001).

[0101] In addition, heart valves from carcinoid heart disease patientsexhibited a marked increase in the number 5HT_(1B) receptor positivecells when compared to number of 5HT_(1B) receptor positive cellsdetected in normal control tricuspid valves.

[0102] To examine the direct proliferative effects of serotonin,cultured porcine aortic valve subendothelial cells were treated with 1μM, 100 nM, and 10 nM of serotonin and then tested using a ³H thymidineincorporation assay. Serotonin treatment markedly increased cellproliferation in the subendothelial cells as compared to untreated cells(33-fold, p<0.001). Incubation with serotonin and a 5HT_(1B) receptorantagonist, methiothepin (7 nM), inhibited the serotonin-inducedproliferation.

[0103] These results indicate that serotonin is a powerful directmitogen on subendothelial valve cells, and that this mitogenic effectis, at least in part, mediated via 5HT1b receptors. Thus, 5HT_(1B)receptor antagonists such as pindolol, cyanopindolol, 5-HT-moduline, andmethiothepin can be used to treat carcinoid heart disease.

[0104] Human valve tissue was also stained with sirius red, which stainscollagen I, as described elsewhere (Dayan et al., Histochem. 93:27-29(1989)). Briefly, slides containing human valve tissue weredeparaffinized and rehydrated through the following solutions: xylenetwice for five minutes, 100% ethanol twice for ten seconds, and 95%ethanol twice for ten seconds. After rehydration, the slides wereincubated for one hour at room temperature in 0.1 percent sirius redsolution. The 0.1 percent sirius red solution contained 0.1 g direct red80 (Sigma, catalog number D0303) in 100 mL of saturated picric acid.After the one hour incubation, the slides were incubated in one percentacetic acid for 30 minutes, rinsed with distilled water for ten secondsthree times, and counterstained with hematoxylin for 20 seconds. The onepercent acetic acid contained one mL of glacial acetic acid in 99 mLdistilled water. After counterstaining, the slides were treated withacid alcohol, rinsed with tap water, dehydrated through alcohols,cleared in xylene, and mounted. The acid alcohol contained one mLhydrochloric acid, 29 mL distilled water, and 70 mL 100% ethanol.

[0105] Sirius red staining revealed a qualitative increase in collagen Istaining in carcinoid heart valve tissue as compared to normal heartvalve tissue.

Other Embodiments

[0106] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A non-murine heart valve cell containing anexogenous nucleic acid that encodes a polypeptide having nitric oxidesynthase activity.
 2. The cell of claim 1, wherein said cell is anendothelial cell.
 3. The cell of claim 1, wherein said cell is amyocyte.
 4. The cell of claim 1, wherein said polypeptide is endothelialnitric oxide synthase.
 5. An isolated heart valve cusp, wherein a cellof said cusp contains an exogenous nucleic acid that encodes apolypeptide having nitric oxide synthase activity.
 6. The cusp of claim5, wherein said cell is porcine.
 7. The cusp of claim 5, wherein saidcell is human.
 8. The cusp of claim 5, wherein said polypeptide isendothelial nitric oxide synthase.
 9. A method for making abioprosthetic heart valve, said method comprising: a) obtaining a heartvalve cusp, and b) introducing nucleic acid into a cell of said cusp,wherein said nucleic acid encodes a polypeptide having nitric oxidesynthase activity.
 10. The method of claim 9, wherein said cell isporcine.
 11. The method of claim 9, wherein said cell is human.
 12. Themethod of claim 9, wherein said polypeptide is endothelial nitric oxidesynthase.
 13. The method of claim 9, wherein said nucleic acid isintroduced into said cell via adenoviral-mediated nucleic acid transfer.14. The method of claim 9, wherein said nucleic acid integrates into thegenome of said cell.
 15. The method of claim 9, wherein said methodcomprises fixing said cusp.
 16. The method of claim 15, wherein saidfixation step occurs after said introduction step.
 17. The method ofclaim 9, wherein said method comprises freezing said cusp.
 18. Themethod of claim 17, wherein said freezing step occurs after saidintroduction step.
 19. A method for slowing the degeneration of a heartvalve within a non-murine mammal, said method comprising introducingnucleic acid encoding a polypeptide having nitric oxide synthaseactivity into a cell of said heart valve such that said polypeptide isexpressed.
 20. The method of claim 19, wherein said introduction step isperformed in vitro.
 21. The method of claim 19, wherein said heart valveis an autograft.
 22. The method of claim 19, wherein said heart valve isan allograft.
 23. The method of claim 19, wherein said heart valve is axenograft.
 24. The method of claim 19, wherein said method comprisesadministering an inhibitor of hydroxymethylglutaryl coA reductaseactivity to said mammal.
 25. The method of claim 24, wherein saidinhibitor comprises pravastatin, atorvastatin, simvastatin, orlovastatin.
 26. A method for slowing heart valve degeneration, saidmethod comprising: a) identifying a mammal at risk of developing heartvalve degeneration, and b) administering an inhibitor ofhydroxymethylglutaryl coA reductase activity to said mammal.
 27. Themethod of claim 26, wherein said mammal contains a heart valvereplacement.
 28. The method of claim 26, wherein said mammal hascongenital valvular disease.
 29. The method of claim 26, wherein saidmammal has bicuspid valvular disease.
 30. A method for treatingcarcinoid heart disease in a mammal, said method comprisingadministering a serotonin receptor antagonist to said mammal.
 31. Themethod of claim 30, wherein said antagonist is specific for a 5HT_(1B)receptor.
 32. The method of claim 30, wherein said antagonist comprisesaβ-blocker.
 33. The method of claim 30, wherein said antagonist ispindolol.
 34. A method for identifying an inhibitor of heart valvedegeneration, said method comprising: a) contacting heart valve cellswith a stimulant such that said cells proliferate, b) contacting saidcells with a test compound, and c) determining if said test compoundreduced the proliferation of said cells, wherein the reduction ofproliferation indicates that said test compound is an inhibitor of heartvalve degeneration.
 35. A method for determining the safety of a drug,said method comprising: a) contacting heart valve cells with said drug,and b) determining if said drug induced proliferation of said cells,wherein the induction of proliferation indicates that said drug promotesheart valve degeneration.